Freshly shed horse chestnut (A. hippocastanum) seeds were collected from the Main Botanical Garden (Moscow, Russia) in October 2007–2009. They were stratified at 4 °C in wet sand for 4 months to break dormancy. During the course of stratification, seeds were regularly examined for radicle emergence under optimum water supply and temperature (27 °C) in the dark. In this way, we were able to distinguish between deep dormancy, dormancy release and non-dormant state. Initiation of germination, i.e. radicle emergence, was evaluated as the appearance of a 1-mm-long radicle tip. During stratification, excised embryonic axes were fixed for microscopical examination and also stored at –20 °C for later biochemical analysis.
Water content was routinely measured in embryonic axes by weighing before and after drying. Vital staining of vacuoles in longitudinal sections of freshly excised embryonic axes was performed with 0.01 % Neutral Red (Baryckina et al. 2004
). Sections 60 µm thick were cut with an HM-650V vibratome and examined under an Axio Imager D1 microscope (Carl Zeiss, Oberhofen, Germany). For light microscopy, embryonic axes were fixed according to Birch-Girschfeld with a Zenker fixative modified for vacuole preservation. The fixative consisted of 3 g of HgCl2
, 2.5 g of K2
and 1 g of Na2
per 100 mL of H2
O. Just prior to fixation, 5 mL of 10 % aqueous formaldehyde were added. After 24 h fixation, axes were washed in running water and transferred to I2
+KI solution to remove residual HgCl2
. The material was then dehydrated and embedded in paraffin. Longitudinal sections were stained with Procion Bright Blue RS and Procion Bright Red 2BS dyes according to Ivanov and Litinskaya (Ivanov 1987
). The size and number of cells in radicles and hypocotyls were measured under a light microscope in the third longitudinal rows of the outer cortex.
To characterize membrane proteins, microsomal fractions from embryonic axes were used. The axes were first excised from intact seeds during stratification and then either immediately frozen or cultivated to radicle emergence in water in Petri dishes in the dark at 27 °C. Frozen axes weighing 15 g (300 embryonic axes from dormant seeds or 150 protruding axes) were homogenized at 4 °C in extraction buffer comprising 300 mM sucrose, 100 mM Tris-HCl (pH 8.0), 10 mM ethylenediaminetetraacetic acid, 5 mM potassium meta-bisulphite, 5 mM dithiothreitol (DTT), 5 mM phenylmethylsulphonyl fluoride, 0.6 % polyvinylpyrollidone in the ratio of 1 : 20. The microsomal fraction was sedimented by centrifugation for 1 h at 100 000 g
, and the pellet resuspended in the same medium. Prior to electrophoresis, microsomal preparations were transferred to two-fold buffer, containing 0.125 M Tris-HCl (pH 6.8), 4 % sodium dodecyl sulphate (SDS), 20 % glycerol, 200 mM DTT and 0.02 % bromophenol blue. Proteins were then separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis with a Midget Electrophoresis Unit 2050 (LKB, Bromma, Sweden) according to Laemmli (1970)
. Gels were stained with Coomassie R-250. Molecular masses were determined with protein markers (10–225 kDa) from Promega (Madison, WI, USA).
For western blot analysis, electrophoretically separated proteins were transferred onto nitrocellulose membranes (Sigma, St Louis, MO, USA) using a Multiphor Electrophoresis Unit (LKB, Sweden). The membrane was washed three times for 5 min with a buffer containing 10 mM phosphate buffer (pH 7.4), 2.7 mM KCl, 137 mM NaCl, 0.05 % Tween 20 (PBS-T) and blocked with 5 % non-fat dry milk in PBS-T.
To identify plasmalemma (PIP) and tonoplast (TIP) aquaporins as well as vacuolar H+-ATPase, nitrocellulose membranes were treated with appropriate antibodies. Nitrocellulose blots were treated with anti-PIP1;1, anti-PIP2;2 and anti-TIP2 antibodies kindly provided by Professor C. Maurel (École supérieure agronomique, Montpellier, France). Antibodies against TIP3;1 were produced in our institute, and the antibodies against subunit E of plant vacuolar H+-ATPase from Arabidopsis thaliana were purchased from Antisera (Vannas, Sweden). Protein bands were visualized with secondary antibodies coupled with horseradish peroxidase (Promega).
The open or closed state of water channels in the membranes was assessed by a method previously developed for growing roots (Barrouclough et al. 2000
; Javot and Maurel 2002
). This involves measuring water absorption, its inhibition by mercuric chloride and subsequent restoration by certain reductants such as DTT. Water uptake by control embryonic axes was measured as an increase in fresh weight. Other embryonic axes were weighed and transferred to 0.5 mM mercuric chloride for 30 min, then weighed again to evaluate the inhibition of water inflow by Hg2+
ions. The rinsed embryonic axes were then incubated in 10 mM DTT to eliminate the mercury-induced inhibition of water uptake and weighed again.
Vacuolar acid invertase activity was measured after protein extraction with 0.01 M phosphate buffer, pH 6.5, followed by centrifugation and subsequent supernatant dialysis against the same buffer for 24 h. The reaction was carried out in 0.01 M phosphate-citrate buffer, pH 5.5, with the substrates (50 mM sucrose or raffinose) at 30 °C for 40 min. Fructose content was estimated by colour reaction with resorcinol at 520 nm with a Genesys 10uv spectrophotometer (Thermo, Madison, WI, USA). To characterize the molecular properties of acid vacuolar invertase, native electrophoresis in 7 % polyacrylamide gels (Davis 1964
) was used with subsequent invertase identification. Protein separation was carried out for 2.5 h at 20 mA, the gel was washed in 0.01 M phosphate-citrate buffer, pH 5.5, for 10 min at 35 °C in darkness and transferred to fresh buffer containing 0.6 M sucrose for an hour at 37 °C in darkness. The invertase reaction was terminated by boiling in 4 % NaOH containing 0.2 % 2,3,5-triphenyltetrazolium chloride. The red colour produced by fructose formed indicated the location of invertase protein within the gel.
Total protein was estimated with a Sigma BCA protein assay. The colour reaction was measured at 620 nm with a Genesys 10uv spectrophotometer (Thermo) with bovine serum albumin as the standard.